Towards two-photon excited endogenous fluorescence lifetime imaging microendoscopy.

In situ fluorescence lifetime imaging microscopy (FLIM) in an endoscopic configuration of the endogenous biomarker nicotinamide adenine dinucleotide (NADH) has a great potential for malignant tissue diagnosis. Moreover, two-photon nonlinear excitation provides intrinsic optical sectioning along with enhanced imaging depth. We demonstrate, for the first time to our knowledge, nonlinear endogenous FLIM in a fibered microscope with proximal detection, applied to NADH in cultured cells, as a first step to a nonlinear endomicroscope, using a double-clad microstructured fiber with convenient fiber length (> 3 m) and excitation pulse duration (≈50 fs). Fluorescence photons are collected by the fiber inner cladding and we show that its contribution to the impulse response function (IRF), which originates from its intermodal and chromatic dispersions, is small (< 600 ps) and stable for lengths up to 8 m and allows for short lifetime measurements. We use the phasor representation as a quick visualization tool adapted to the endoscopy speed requirements.

Generalization of the polar representation in time domain fluorescence lifetime imaging microscopy for biological applications: practical implementation.

The polar representation or phasor, which provides a fast and visual indication on the number of exponentials present in the intensity decay of the fluorescence lifetime images is increasingly used in time domain fluorescence lifetime imaging microscopy experiments. The calculations of the polar coordinates in time domain fluorescence lifetime imaging microscopy experiments involve several experimental parameters (e.g. instrumental response function, background, angular frequency, number of temporal channels) whose role has not been exhaustively investigated. Here, we study theoretically, computationally and experimentally the influence of each parameter on the polar calculations and suggest parameter optimization for minimizing errors. We identify several sources of mistakes that may occur in the calculations of the polar coordinates and propose adapted corrections to compensate for them. For instance, we demonstrate that the numerical integration method employed for integrals calculations may induce errors when the number of temporal channels is low. We report theoretical generalized expressions to compensate for these deviations and conserve the semicircle integrity, facilitating the comparison between fluorescence lifetime imaging microscopy images acquired with distinct channels number. These theoretical generalized expressions were finally corroborated with both Monte Carlo simulations and experiments.

Quantitative comparison of polar approach versus fitting method in time domain FLIM image analysis.

We calculate here analytically the performance of the polar approach (or phasor) in terms of signal-to-noise ratio and F values when performing time-domain Fluorescence Lifetime Imaging Microscopy (FLIM) to determine the minimal number of photons necessary for FLIM measurements (which is directly related to the F value), and compare them to those obtained from a well-known fitting strategy using the Least Square Method (LSM). The importance of the fluorescence background on the lifetime measurement precision is also investigated. We demonstrate here that the LSM does not provide the best estimator of the lifetime parameter for fluorophores exhibiting mono-exponential intensity decays as soon as fluorescence background is superior to 5%. The polar approach enables indeed to determine more precisely the lifetime values for a limited range corresponding to usually encountered fluorescence lifetime values. These theoretical results are corroborated with Monte Carlo simulations. We finally demonstrate experimentally that the polar approach allows distinguishing in living cells two fluorophores undetectable with usual time-domain LSM fitting software.

Control of pulse-to-pulse fluctuations in visible supercontinuum.

Long-pulse supercontinuum sources are initiated by modulation instability and consequently suffer from stochastic shot-to-shot variations of their spectral power density. In this paper, we provide a measurement of pulse-to-pulse fluctuations over the whole supercontinuum spectrum, and we show that their spectral dependence follows the group index curve of the fiber. Then, we demonstrate a significant reduction of supercontinuum pulse-to-pulse fluctuations in the visible by using a photonic crystal fiber with longitudinally tailored guidance properties. We finally show numerically that this new source would allow a significant improvement of the signal-to-noise ratio in fluorescence microscopy.

Improvement of FISH mapping resolution on combed DNA molecules by iterative constrained deconvolution: a quantitative study.

Image restoration approaches, such as digital deconvolution, are becoming widely used for improving the quality of microscopic images. However, no quantification of the gain in resolution of fluorescence images is available. We show that, after iterative constrained deconvolution, fluorescent cosmid signals appear to be 25% smaller, and 1.2-kb fragment signals on combed molecules faithfully display the expected length.

Multiple links between the NuA4 histone acetyltransferase complex and epigenetic control of transcription.

NuA4 is an essential histone H4/H2A acetyltransferase complex that interacts with activators and stimulates transcription in vitro. We have identified three novel NuA4 subunits: Act3/Arp4, an actin-related protein implicated in epigenetic control of transcription, Act1, and Epl1, a protein homologous to Drosophila Enhancer of Polycomb. Act3/Arp4 binds nucleosomes in vitro and is required for NuA4 integrity in vivo. Mutations in ACT3 and acetyltransferase-encoding ESA1 cause gene-specific transcription defects. Accordingly, NuA4 is localized in precise loci within the nucleus and does not overlap with the silent chromatin marker Sir3. These data along with the known epigenetic roles of Act3/Arp4 and homologs of Epl1 and Esa1 strongly support an essential role for chromatin structure modification by NuA4 in transcription regulation in vivo.

Nonrandom distribution of metaphase AgNOR staining patterns on human acrocentric chromosomes.

The metaphase nucleolar organizer regions (NORs) contain ribosomal genes associated with proteins such as upstream binding factor (UBF) and RNA polymerase I (RPI). These genes are clustered in 10 loci of the human acrocentric chromosomes (13, 14, 15, 21, and 22). Some NOR-associated proteins, termed AgNOR proteins, can be specifically stained by silver. In this study we took advantage of technical advances in digital imaging, image restoration techniques, and factorial correspondence analysis (FCA) to study the different AgNOR staining patterns of metaphase chromosomes in human lymphocytes. Three predominant patterns could be distinguished: pair (47%), stick-like (28%), and unstained (18%) structures. By studying the frequency of occurrence of each pattern on different chromosomes, two groups could be defined. Chromosomes 13, 14, and 21 carried predominantly pair or stick-like AgNOR structures, whereas chromosomes 15 and 22 mainly carried pair AgNOR structures or remained unstained. We suggest that the different AgNOR shapes reflect both the number of ribosomal genes carried by each chromosome and the differential recruitment of active ribosomal genes in each NOR cluster. This is the first study showing a nonrandom distribution of AgNOR shape among acrocentric chromosomes.

3-D organization of ribosomal transcription units after DRB inhibition of RNA polymerase II transcription.

In each bead of the nucleolar necklace, using adenosine analog DRB-treated PtK1 cells, we investigated the three components of rDNA transcription, i.e. the gene, transcription factor UBF and transcripts. In situ hybridization revealed the unraveling and 3-D dispersion of most of the rDNA coding sequences within the nucleus. The signals were small, of similar intensity and tandemly organized in the necklace. This observation is compatible with the fact that they might correspond to single gene units. Active transcription was visualized in these units, demonstrating that they were active functional units. Transcript labeling was not similar for each unit, contrary to UBF labeling. UBF and rRNA transcripts were only partially colocalized, as demonstrated by 3-D image analysis and quantification. As visualized by electron microscopy, the necklace was composed of a small fibrillar center partially surrounded by a dense fibrillar component. The 3-D arrangement of this individual unit in the necklace, investigated both by confocal and electron microscopy in the same cells, showed that the individual beads were linked by a dense fibrillar component. The reversibility of this organization after removal of DRB indicated that the beads in the necklace are certainly the elementary functional domain of the nucleolus. In addition, these results lead us to suggest that the organization of a functional domain, presumably corresponding to a single gene, can be studied by in situ approaches.

S-phase-dependent action of cycloheximide in relieving chromatin-mediated general transcriptional repression.

Chromatin plays a major role in the tight regulation of gene expression and in constraining inappropriate gene activity. Replication-coupled chromatin assembly ensures maintenance of these functions of chromatin during S phase of the cell cycle. Thus treatment of cells with an inhibitor of translation, such as cycloheximide (CX), would be expected to have a dramatic effect on chromatin structure and function, essentially in S phase of the cell cycle, due to uncoupled DNA replication and chromatin assembly. In this work, we confirm this hypothesis and show that CX can induce a dramatic S-phase-dependent alteration in chromatin structure that is associated with general RNA polymerase II-dependent transcriptional activation. Using two specific RNA polymerase II-transcribed genes, we confirm the above conclusion and show that CX-mediated transcriptional activation is enhanced during the DNA replication phase of the cell cycle. Moreover, we show co-operation between an inhibitor of histone deacetylase and CX in inducing gene expression, which is again S-phase-dependent. The modest effect of CX in inducing the activity of a transiently transfected promoter shows that the presence of the promoter in an endogenous chromatin context is necessary in order to observe transcriptional activation. We therefore suggest that the uncoupled DNA replication and histone synthesis that occur after CX treatment induces a general modification of chromatin structure, and propose that this general disorganization of chromatin structure is responsible for a widespread activation of RNA polymerase II-mediated gene transcription.

The three-dimensional study of chromosomes and upstream binding factor-immunolabeled nucleolar organizer regions demonstrates their nonrandom spatial arrangement during mitosis.

The volumic rearrangement of both chromosomes and immunolabeled upstream binding factor in entire well-preserved mitotic cells was studied by confocal microscopy. By using high-quality three-dimensional visualization and tomography, it was possible to investigate interactively the volumic organization of chromosome sets and to focus on their internal characteristics. More particularly, this study demonstrates the nonrandom positioning of metaphase chromosomes bearing nucleolar organizer regions as revealed by their positive upstream binding factor immunolabeling. During the complex morphogenesis of the progeny nuclei from anaphase to late telophase, the equal partitioning of the nucleolar organizer regions is demonstrated by quantification, and their typical nonrandom central positioning within the chromosome sets is revealed.

Electron tomography of metaphase nucleolar organizer regions: evidence for a twisted-loop organization.

Metaphase nucleolar organizer regions (NORs), one of four types of chromosome bands, are located on human acrocentric chromosomes. They contain r-chromatin, i.e., ribosomal genes complexed with proteins such as upstream binding factor and RNA polymerase I, which are argyrophilic NOR proteins. Immunocytochemical and cytochemical labelings of these proteins were used to reveal r-chromatin in situ and to investigate its spatial organization within NORs by confocal microscopy and by electron tomography. For each labeling, confocal microscopy revealed small and large double-spotted NORs and crescent-shaped NORs. Their internal three-dimensional (3D) organization was studied by using electron tomography on specifically silver-stained NORs. The 3D reconstructions allow us to conclude that the argyrophilic NOR proteins are grouped as a fiber of 60-80 nm in diameter that constitutes either one part of a turn or two or three turns of a helix within small and large double-spotted NORs, respectively. Within crescent-shaped NORs, virtual slices reveal that the fiber constitutes several longitudinally twisted loops, grouped as two helical 250- to 300-nm coils, each centered on a nonargyrophilic axis of condensed chromatin. We propose a model of the 3D organization of r-chromatin within elongated NORs, in which loops are twisted and bent to constitute one basic chromatid coil.

Applications of medium-voltage STEM for the 3-D study of organelles within very thick sections.

Scanning transmission electron microscopy at 300kV enables the visualization of nucleolar silver-stained structures within thick sections (3-8 microns) of Epon-embedded cells at high tilt angles (-50 degrees; +50 degrees). Thick sections coated with gold particles were used to determine the best conditions for obtaining images with high contrast and good resolution. For a 6-microns-thick section the values of thinning and shrinkage under the beam are 35 to 10%, respectively. At the electron density used in these experiments (100e-/A2/s) it is estimated that these modifications of the section stabilized in less than 10 min. The broadening of the beam through the section was measured and calculations indicated that the subsequent resolution reached 100 nm for objects localized near the lower side of 4-microns-thick sections with a spot-size of 5.6 nm. Comparing the same biological samples, viewed alternately in CTEM and STEM, demonstrated that images obtained in STEM have a better resolution and contrast for sections thicker than 3 microns. Therefore, the visualization of densely stained structures, observed through very thick sections in the STEM mode, will be very useful in the near future for microtomographic reconstruction of cellular organelles.